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Mob/oriT,一种用于苏云金芽孢杆菌和蜡样芽孢杆菌中无标记基因操作的可移动位点特异性重组系统。

Mob/oriT, a mobilizable site-specific recombination system for unmarked genetic manipulation in Bacillus thuringiensis and Bacillus cereus.

作者信息

Wang Pengxia, Zhu Yiguang, Zhang Yuyang, Zhang Chunyi, Xu Jianyi, Deng Yun, Peng Donghai, Ruan Lifang, Sun Ming

机构信息

State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, People's Republic of China.

出版信息

Microb Cell Fact. 2016 Jun 10;15(1):108. doi: 10.1186/s12934-016-0492-9.

Abstract

BACKGROUND

Bacillus thuringiensis and Bacillus cereus are two important species in B. cereus group. The intensive study of these strains at the molecular level and construction of genetically modified bacteria requires the development of efficient genetic tools. To insert genes into or delete genes from bacterial chromosomes, marker-less manipulation methods were employed.

RESULTS

We present a novel genetic manipulation method for B. thuringiensis and B. cereus strains that does not leave selection markers. Our approach takes advantage of the relaxase Mob02281 encoded by plasmid pBMB0228 from Bacillus thuringiensis. In addition to its mobilization function, this Mob protein can mediate recombination between oriT sites. The Mob02281 mobilization module was associated with a spectinomycin-resistance gene to form a Mob-Spc cassette, which was flanked by the core 24-bp oriT sequences from pBMB0228. A strain in which the wild-type chromosome was replaced with the modified copy containing the Mob-Spc cassette at the target locus was obtained via homologous recombination. Thus, the spectinomycin-resistance gene can be used to screen for Mob-Spc cassette integration mutants. Recombination between the two oriT sequences mediated by Mob02281, encoded by the Mob-Spc cassette, resulted in the excision of the Mob-Spc cassette, producing the desired chromosomal alteration without introducing unwanted selection markers. We used this system to generate an in-frame deletion of a target gene in B. thuringiensis as well as a gene located in an operon of B. cereus. Moreover, we demonstrated that this system can be used to introduce a single gene or an expression cassette of interest in B. thuringiensis.

CONCLUSION

The Mob/oriT recombination system provides an efficient method for unmarked genetic manipulation and for constructing genetically modified bacteria of B. thuringiensis and B. cereus. Our method extends the available genetic tools for B. thuringiensis and B. cereus strains.

摘要

背景

苏云金芽孢杆菌和蜡样芽孢杆菌是蜡样芽孢杆菌属中的两个重要菌种。在分子水平上对这些菌株进行深入研究以及构建转基因细菌需要开发高效的遗传工具。为了将基因插入细菌染色体或从细菌染色体中删除基因,采用了无标记操作方法。

结果

我们提出了一种针对苏云金芽孢杆菌和蜡样芽孢杆菌菌株的新型遗传操作方法,该方法不会留下选择标记。我们的方法利用了苏云金芽孢杆菌质粒pBMB0228编码的松弛酶Mob02281。除了其转移功能外,这种Mob蛋白还可以介导oriT位点之间的重组。Mob02281转移模块与一个壮观霉素抗性基因相连,形成一个Mob-Spc盒,其两侧是来自pBMB0228的核心24bp oriT序列。通过同源重组获得了一个菌株,其中野生型染色体在目标位点被含有Mob-Spc盒的修饰拷贝所取代。因此,壮观霉素抗性基因可用于筛选Mob-Spc盒整合突变体。由Mob-Spc盒编码的Mob02281介导的两个oriT序列之间的重组导致Mob-Spc盒的切除,产生所需的染色体改变,而不会引入不需要的选择标记。我们使用这个系统在苏云金芽孢杆菌中产生了一个目标基因的框内缺失,以及在蜡样芽孢杆菌一个操纵子中的一个基因的框内缺失。此外,我们证明了这个系统可用于在苏云金芽孢杆菌中引入单个基因或感兴趣的表达盒。

结论

Mob/oriT重组系统为苏云金芽孢杆菌和蜡样芽孢杆菌的无标记遗传操作和构建转基因细菌提供了一种有效的方法。我们的方法扩展了苏云金芽孢杆菌和蜡样芽孢杆菌菌株可用的遗传工具。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/245a/4902927/1e4e7c99adad/12934_2016_492_Fig1_HTML.jpg

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